3D multifocus astigmatism and compressed sensing (3D MACS) based superresolution reconstruction

Abstract

Abstract:Single molecule based superresolution techniques (STOR-M/PALM) achieve nanometer spatial resolution by integrating the temporal information of the switching dynamics of fluorophores (emitters). When emitter density is low for each frame, they are located to the nanometer resolution. However, when the emitter density rises, causing significant overlapping, it becomes increasingly difficult to accurately locate individual emitters. This is particularly apparent in three dimensional (3D) localization because of the large effective volume of the 3D point spread function (PSF). The inability to precisely locate the emitters at a high density causes poor temporal resolution of localization-based superresolution technique and significantly limits its application in 3D live cell imaging. To address this problem, we developed a 3D high-density superresolution imaging platform that allows us to precisely locate the positions of emitters, even when they are significantly overlapped in three dimensional space. Our platform involves a multi-focus system in combination with astigmatic optics and an 1 -Homotopy optimization procedure. To reduce the intrinsic bias introduced by the discrete formulation of compressed sensing, we introduced a debiasing step followed by a 3D weighted centroid procedure, which not only increases the localization accuracy, but also increases the computation speed of image reconstruction. We implemented our algorithms on a graphic processing unit (GPU), which speeds up processing 10 times compared with central processing unit (CPU) implementation. We tested our method with both simulated data and experimental data of fluorescently labeled microtubules and were able to reconstruct a 3D microtubule image with 1000 frames (512×512) acquired within 20 seconds.

Roles of the fission yeast UNC-13/Munc13 protein Ync13 in late stages of cytokinesis

“…Usually we took 4500–8000 images for each field. Superresolution images were reconstructed using Memp-STORM ( Huang, Sun, et al. , 2015 ).…”

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…Incorporating astigmatic PSF with quantum dot blinking, the 3D distributions of the plasma membranes and internal epidermal growth factor receptor molecules in breast cancer cells can be identified with lateral and axial resolution of 8–17 nm and 60–80 nm, respectively . Furthermore, by combining MPM with astigmatic PSF, 3D super‐resolution image reconstruction with high labeling density and large image depth can be performed …”

“…[227] Furthermore, by combining MPM with astigmatic PSF, 3D super-resolution image reconstruction with high labeling density and large image depth can be performed. [228][229][230] However, introduction of astigmatism to the PSF generates certain problems; for example, the cylindrical lens induces optical aberrations that degrade the localization precision and resolution. This particular problem can be corrected using deformable mirrors to obtain an axial localization precision of 20-40 nm.…”

“…Although the imaging performance of these localization-based axial SRFM methods is affected by many experimental factors, such as the marker labeling density, detector noise, optical system aberrations, refractive index mismatch, photon efficiency, and field-dependent aberrations, it is possible to design the optimal PSF with few localization errors by considering the PSF as individual designing parameter, and analyzing and balancing the interactions among the pupil function, ambiguity function, Fisher information, CRLB, and the relevant experimental parameters mentioned above. [255,256,261] Benefiting from the development of 3D localization-based super-resolution reconstruction algorithms, [228,229,262,263] these PSF engineering techniques have become a powerful research tool for many challenging topics, being used for velocity measurement, [217] dipole orientation analysis, [264] spectral traits tracking, [222] etc. More recently, Gustavsson et al demonstrated the value of PSF engineering with a long axial range coupled with tilted light sheet illumination to produce impressive cellular super-resolution data.…”

Advances in 3D single particle localization microscopy

“…101,104 CS makes no such assumptions and has therefore been demonstrated to work with very highdensity images. [105][106][107][108] Figure 12 shows a comparison between typical maximum densities of LS and CS algorithms when used with the DH-PSF. 106…”